This invention relates to an antithrombosis enzyme derived from the snake venom of an acutus species.
Anti-thrombus drugs extracted from acutus venom have been reported in the literature, e.g., xe2x80x9cPreparation and Study of Anti-thrombus Enzymes No. 1, 2, and 3xe2x80x9d, Journal of the Medical Univ. of China, 1989.18 (special issue); and xe2x80x9cTechnique for Extracting Definriogenase from the venom of Agkistrodon acutus,xe2x80x9d CN 92102645.5 (CN 1065680.A). These anti-thrombus drugs are proteinase components extracted from the snake venom. They act like thrombase with hemorrhagic side-effect. In addition, some of these products are not single component proteinase, but a mixture of different components, which limits the pharmaceutical application of these drugs in human.
Other snake venom derived pharmaceutical products include Ancrod, Trigtamin and Integrilin (see Matsuzaki et al., Biochem. Biophy. Res. Com. 220(2):382-387, 1996; Morita et al., Natural Toxins II, pp187-196, Edited by B. R. Singh and A. T. Tu, Plenum Press, New York, 1996; U.S. Pat. Nos. 5,196,403, 5,242,810, 5,453,370, 4,017,012, 5,344,783, 5,686,571, 5,523,292, 5,066,592 and 5,342,830).
Within the scope of this invention, Applicant has extracted, purified and cloned an antithrombosis enzyme (ATE, also called a fibrinolytic enzyme in the provisional application) from the venom of Southern-Anhui Agkistrodon acutus in China. This enzyme degrades both fibrinogen and fibrin, and inhibits platelet aggregation. It is useful for preventing and treating vaso-occulusive and thromboembolic disorders, including, but not limited to, myocardial infarction, restenosis, peripheral anginaphraxis, angiopathic thrombosis, cerebral thrombosis, ischemic cerebral vascular diseases, unstable angina, acute thrombosis, unstable stenocardia and hemiparalysis caused by cerebral thrombosis.
The present invention provides methods and compositions for preventing or treating diseases and processes mediated (caused or aggravated) by undesired and/or uncontrolled thrombosis by administering to a human or animal a composition containing or capable of expressing the antithrombosis enzyme in a dosage sufficient to prevent, reduce, eliminate or inhibit thrombosis. The antithrombosis enzyme may be substantially purified or in a crude extract. The antithrombosis enzyme may be produced from snake venom, chemically synthesized or expressed from a recombinant vector. It may also be combined with a pharmaceutically acceptable excipient or carrier, and optionally sustained-release compounds or compositions, such as biodegradable polymers, to form therapeutic compositions.
The present invention is particularly useful for treating or preventing acute and recurrent cerebral thrombosis, myocardial infarction, restenosis, peripheral anginaphraxis, angiopathic thrombosis, ischemic cerebral vascular thrombosis, unstable angina, unstable stenocardia, and thromboangitis obliterans. Administration of the antithrombosisi enzyme can prevent blood clot formation and reduce, diminish or dissolve blood clot. The antithrombosis enzyme may also be used in combination with other compositions and procedures for the treatment of thrombosis. For example, it may be used in combination with a thrombolytic agent known in the art, which includes, but is not limited to, tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, anisolated streptokinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above. In these combination compositions, the antithrombosis enzyme and other thrombolytic agent work in a complementary fashion to dissolve blood clots, resulting in decreased reperfusion times and increased reocclusion times in patients treated with them. The use of the antithrombosis enzyme in the compositions of this invention advantageously allows the administration of a thrombolytic reagent in dosages previously considered too low to result in thrombolytic effects if given alone. This avoids some of the undesirable side effects associated with the use of thrombolytic S agents, such as bleeding complications. The compositions of this invention may also be used before, concurrent with, or after angioplastic or fibrolytic treatment to prevent or treat restenosis.
Thus, in a first aspect, this invention features an isolated, purified or recombinant antithrombosisi enzyme which has (i) a molecular weight of between about 28 kD and about 32 kD when analyzed by polyacrylamide gel electrophoresis, (ii) an aspartic acid content of between about 2% and about 5%, and (iii) a glutamic acid content of between about 2% and about 5%. This enzyme has the ability to hydrolyze fibrin, dissolve thrombus, inhibit platelet aggregation, and inhibit the formation of thrombus.
In a preferred embodiment, the enzyme has fibrinolytic activity of no less than one fibrinolytic activity unit per mg protein. In another preferred embodiment, the enzyme has fibrinolytic activity of between about one and about three fibrinolytic activity units per mg protein. This enzyme specifically hydrolyzes the A (xcex1) chain of fibrinogen. This enzyme completely or almost completely inhibits human platelet aggregation induced by agonists such as ADP, Epinephrine, Thrombin and collagen. This enzyme has no detectable hydrolysis effect on casein. The enzyme dissolves arterial and venous thrombus in a mammal, prevent thrombosis, reduce blood viscosity, and improve microcirculation. At the same time, this enzyme has minimum effect on the thromosystem, resulting in little possibility of hemorrhage. This enzyme is different from related enzymes from other Acutus species (e.g., IX/X binding proteins, Matsuzaki et al., Biochem. Biophy. Res. Com. 220(2):382-387, 1996; Morita et al., Natural Toxins II, pp187-196, Edited by B. R. Singh and A. T. Tu, Plenum Press, New York, 1996) in that this enzyme has both fibrinolytic activity and antiplatelet aggregation activity, and less hemorrhagic activity.
In other preferred embodiments, this enzyme is purified from Southern-Anhui Agkistrodon acutus. The enzyme is a heterodimer of A chain and B chain each with a molecular weight of about 14 KD to about 16 KD. The A chain has at its amino end the following sequence Sequence ID No. 3:
Asp-Cys-Ser-Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Lys-Gln-Ser-Lys-Thr-Trp-Thr-Asp-Ala-Glu-Ser-Phe-, and the B chain has at its amino end the following sequence Sequence ID No. 4:
Asp-Cys-Pro-Ser-Glu-Trp-Ser-Ser-Tyr-Glu-Gly-Phe-Cys-Tyr-Lys-Pro-Phe-. Preferrably, the A chain and the B chain are linked by one or more disulfide bond.
In other preferred embodiments, this antithrombosis enzyme contains Ca++ and/or has aspartic acid at its amino terminus.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polypeptide isolated from a natural source or synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring amino acid sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is the predominate sequence present (at least 10-20% more than any other sequence) and is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantlyxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The amino acid from other sources may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid.
By xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
By xe2x80x9crecombinantxe2x80x9d is meant a polypeptide or enzyme produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. This invention features recombinant ATE and its fragments obtainable using techniques known to those skilled in the art, including those described in McDonnell et al., U.S. patent application Ser. No. 08/223,943 filed Apr. 6, 1994, Evans et al., U.S. Pat. No. 5,071,773, and PCT application, PCT/US91/00399 filed Jan. 22, 1991 (International Publication No. WO 91/12258), incorporated by reference herein.
In a second aspect, this invention features isolated, purified or recombinant polypeptide fragments of the A chain and the B chain of the antithrombosis enzyme. Preferably, these fragments contain no less than 15, 20, 30 or 40 contiguous amino acid residues from the A or B chain. For example, these fragments may contain no less than 15, 20, 30 or 40 contiguous amino acid residues from SEQ ID NO: 2. Such polypeptide fragments can be synthesized chemically or expressed from recombinant vectors. They are useful for generating monoclonal antibodies which bind to both the polypeptide fragments and the intact antithrombosis enzyme (see U.S. Pat. Nos. 5,733,738, 5,015,571, incorporated by reference herein). Monoclonal antibodies so generated can be attached to solid support and used to purify the antithrombosis enzyme from crude venom extract or cell extract by affinity chromatography.
The recombinant polypeptide fragments of the A chain and the B chain can be expressed from recombinant nucleic acid encoding such polypeptide fragments. For example, polypeptide fragments of the A chain can be expressed from recombinant nucleic acid containing no less than 45, 60, 90 or 120 contiguous nucleotides from SEQ ID NO: 1 or its fully complementary strand of the same length and a promoter effective to initiate transcription of the contiguous nucleotides in a host cell.
In yet another aspect the invention features an isolated, enriched, or purified antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to the antithrombosis enzyme or a fragment thereof. The antibody contains a sequence of amino acids that is able to specifically bind to the antithrombosis enzyme. The antibody may be prepared with techniques known to those skilled in the art, including, but not limited to, those disclosed in Niman, PCT application PCT/US88/03921 (International Publication No. WO 89/04489), incorporated by reference herein. By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody will bind to the ATE in a certain detectable amount but will not bind other polypeptides to the same extent under identical conditions.
In another aspect the invention features a hybridoma which produces an antibody having specific binding affinity to the antithrombosis enzyme or a fragment thereof. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody.
In another aspect, the invention features an isolated, purified, enriched or purified recombinant nucleic acid encoding the antithrombosis enzyme, a chain of the enzyme, or fragments of the A chain or B chain. For example, the recombinant nucleic acid contains a sequence contiguously encoding SEQ ID NO: 2 and a promoter effective to initiate transcription of the conding sequence in a host cell. In particular, the recombinant nucleic acid contains SEQ ID NO: 1 operably linked to a promoter.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant DNA or RNA isolated from a natural source or synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but does indicate that it is the predominate sequence present (at least 10-20% more than any other nucleotide sequence) and is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it. Therefore, the term does not encompass an isolated chromosome encoding the ATE.
By xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
By xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased in a useful manner and preferably separate from a sequence library. The term xe2x80x9csignificantlyxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The DNA from other sources may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
By xe2x80x9crecombinantxe2x80x9d in reference to a nucleic acid is meant the nucleic acid is produced by recombinant DNA techniques such that it is distinct from a naturally occurring nucleic acid.
By xe2x80x9ccomprisingxe2x80x9d is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
The invention also features a nucleic acid probe for the detection of a nucleic acid encoding the antithrombosis enzyme, its A chain or B chain, or fragments thereof. In an example, the nucleic acid probe contains nucleic acid that will hybridize to SEQ ID NO: 1, but not to the nucleic acid sequence of the IX/X-binding protein (Matsuzaki et al., Biochem. Biophy. Res. Com. 220(2):382-387, 1996) under high stringency hybridization conditions. By xe2x80x9chigh stringency hybridization conditionsxe2x80x9d is meant those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50xc2x0 C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C.; or (3) employ 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5xc3x97 Denhardt""s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2xc3x97SSC and 0.1% SDS. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
In another aspect, the invention describes a recombinant cell or tissue containing an exogenous nucleic acid coding for the antithrombosis enzyme, its A chain or B chain, or fragments thereof.
In another aspect, this invention features a pharmaceutical composition for reducing or eliminating thrombosis in a human or animal subject. This composition contains a pharmaceutically effective amount of the antithrombosis enzyme and a pharmaceutically acceptable carrier.
By xe2x80x9cpharmaceutically effective amountxe2x80x9d is meant an amount of a pharmaceutical compound or composition having a therapeutically relevant effect on thrombosis or diseases or pathological conditions related to thrombosis. A therapeutically relevant effect prevents or relieves to some extent one or more symptoms of thrombosis or diseases or pathological symptoms related to thrombosis in a patient or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of thrombosis or diseases or pathological symptoms related to thrombosis, e.g., reducing or inhibiting thrombosis; curing, reducing, or inhibiting diseases and processes that are mediated by thrombosis.
In another aspect, this invention features a method of reducing or inhibiting thrombosis in a human or animal subject by administering to the subject a pharmaceutically effective amount of the antithrombosis enzyme.
In another aspect, this invention features a method of isolating and/or purifying the antithrombosis enzyme. In this method, a crude extract containing the antithrombosis enzyme, e.g., a crude extract of the snake venom of Southern-Anhui Agkistrodon acutus, is dissolved in a buffer and brought into contact with an anion exchange column, the elution sample with fibrinolytic activity and antiplatelet aggregation activity is collected, concentrated and separated by gel permeation chromatography, and the elution sample with fibrinolytic activity and antiplatelet aggregation activity is collected and desalted. This invention features an antithrombosis enzyme isolated by this process.
The present invention also provides recombinant DNA molecules characterized by a DNA sequence encoding the antithrombosis enzyme alone or fused to a DNA sequence which codes for a conventional anti-thrombosis polypeptide. The synthesis of these DNA molecules may be achieved by methods well known in the art. For example, these recombinant DNA molecules may be isolated from an Agkistrodon acutus venom gland cDNA library. The synthesis of cDNA libraries and the choice of vector into which the cDNA molecules may be cloned are conventional techniques. The invention also relates to hosts transformed with these recombinant DNA molecules, as well as to the recombinant products expressed by these hosts. And the present invention relates to chemically synthesized antithrombosis enzyme. Such synthetic polypeptides may be prepared by conventional chemical synthesis techniques, for example, synthesis on a solid support.
This invention further relates to pharmaceutically acceptable compositions and combinations, and methods utilizing these natural, recombinant or synthetic antiplatelet polypeptides in the treatment extracorporeal blood.
By xe2x80x9cextracorporeal bloodxe2x80x9d is meant blood that is removed from a patient, subjected to extracorporeal treatment, and returned to the patient in processes such as dialysis, blood filtration or blood bypass during surgery. The term also includes blood products which are stored extracorporeally for eventual administration to a patient. Such products include whole blood, platelet concentrates and other blood fractions in which inhibition of platelet aggregation and platelet release is desired.
Other features and advantages of the invention will be apparent from the following drawing and detailed description of the invention and from the claims.